Treatment of bioprosthetic tissues to mitigate post implantation calcification

ABSTRACT

Bioprosthetic tissues are treated by immersing or otherwise contacting fixed, unfixed or partially fixed tissue with a glutaraldehyde solution that has previously been heat-treated or pH adjusted prior to its contact with the tissue. The prior heat treating or pH adjustment of the glutaraldehyde solution causes its free aldehyde concentration to decrease by about 25% or more, preferably by as much as 50%, and allows a “stabilized” glutaraldehyde solution to be obtained at the desired concentration and pH for an optimal fixation of the tissue at high or low temperature. This treatment results in a decrease in the tissue&#39;s propensity to calcify after being implanted within the body of a human or animal patient.

RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 10/992,563, filed Nov. 18, 2004, now abandoned, which is adivisional of U.S. patent application Ser. No. 10/039,367, filed Jan. 3,2002, now U.S. Pat. No. 6,878,168, both of which are expresslyincorporated herein by reference.

FIELD OF THE INVENTION

This invention pertains generally to biomaterials and more particularlyto methods for mitigating the post-implantation calcification ofbioprosthetic materials and the bioprosthetic devices and articlesproduced by such methods.

BACKGROUND OF THE INVENTION

Implantable biological tissues can be formed of human tissues preservedby freezing (i.e., cryopreserving) the so called homograft tissues, orof animal tissues preserved by chemically fixing (i.e., tanning) the socalled bioprosthesis (Carpentier, Biological Tissues in Heart ValveReplacement, Butterworth (1972), Ionescu editor). The type of biologicaltissues used as bioprostheses include cardiac valves, blood vessels,skin, dura mater, pericardium, small intestinal submucosa (“SIStissue”), ligaments and tendons. These biological tissues typicallycontain connective tissue proteins (i.e., collagen and elastin) that actas the supportive framework of the tissue. The pliability or rigidity ofeach biological tissue is largely determined by the relative amounts ofcollagen and elastin present within the tissue and/or by the physicalstructure and configuration of its connective tissue framework. Collagenis the most abundant connective tissue protein present in most tissues.Each collagen molecule is made up of three (3) polypeptide chainsintertwined in a coiled helical configuration.

The techniques used for chemical fixation of biological tissuestypically involve the exposure of the biological tissue to one or morechemical fixatives (i.e., tanning agents) that form cross-linkagesbetween the polypeptide chains within a given collagen molecule (i.e.,intramolecular crosslinkages), or between adjacent collagen molecules(i.e., intermolecular crosslinkages).

Examples of chemical fixative agents that have been utilized tocrosslink collagenous biological tissues include: formaldehyde,glutaraldehyde, dialdehyde starch, hexamethylene diisocyanate andcertain polyepoxy compounds. Of the various chemical fixativesavailable, glutaraldehyde has been the most widely used since thediscovery of its antiimmunological and antidegenerative effects by Dr.Carpentier in 1968. See Carpentier, A., J. Thorac. CardiovascularSurgery, 58: 467-69 (1969). In addition, glutaraldehyde is one of themost efficient sterilization agents. Glutaraldehyde is used as thefixative and the sterilant for many commercially available bioprostheticproducts, such as porcine bioprosthetic heart valves (e.g., theCarpentier-Edwards® stented porcine Bioprosthesis), bovine pericardialheart valves (e.g., Carpentier-Edwards® Pericardial Bioprosthesis) andstentless porcine aortic valves (e.g., Edwards PRIMA Plus® StentlessAortic Bioprosthesis), all manufactured and sold by Edwards LifesciencesLLC, Irvine, Calif.

One problem associated with the implantation of many bioprostheticmaterials is that the connective tissue proteins (i.e., collagen andelastin) within these materials can become calcified followingimplantation within the body. Such calcification can result inundesirable stiffening or degradation of the bioprosthesis. Two (2)types of calcification—intrinsic and extrinsic—are known to occur infixed collagenous bioprostheses. Intrinsic calcification follows theadsorption by the tissue of lipoproteins and calcium binding proteins.Extrinsic calcification follows the adhesion of cells (e.g., platelets)to the bioprosthesis and leads to the development of calcium phosphatecontaining surface plaques on the bioprosthesis.

The factors that affect the rate at which fixed tissue bioprosthesesundergo calcification have not been fully elucidated. However, factorsthought to influence the rate of calcification include the patient'sage, the existence of metabolic disorders (i.e., hypercalcemia,diabetes, etc.), dietary factors, the presence of infection, parenteralcalcium administration, dehydration, in situ distortion of thebioprosthesis (e.g., mechanical stress), inadequate anticoagulationtherapy during the initial period following surgical implantation andimmunologic host-tissue responses.

Various techniques have heretofore been proposed for mitigating the insitu calcification of glutaraldehyde-fixed bioprostheses or forotherwise improving the glutaraldehyde fixation process. Included amongthese are the methods described in U.S. Pat. No. 4,729,139 (Nashef)entitled Selective Incorporation of a Polymer into ImplantableBiological Tissue to Inhibit Calcification; U.S. Pat. No. 4,885,005(Nashef et al.) entitled Surfactant Treatment of Implantable BiologicalTissue To Inhibit Calcification; U.S. Pat. No. 4,648,881 (Carpentier etal.) entitled Implantable Biological Tissue and Process For PreparationThereof; U.S. Pat. No. 4,976,733 (Girardot) entitled Prevention ofProsthesis Calcification; U.S. Pat. No. 4,120,649 (Schechter) entitledTransplants; U.S. Pat. No. 5,002,566 (Carpentier) entitled CalcificationMitigation of Bioprosthetic Implants; EP 103947A2 (Pollock et al.)entitled Method For Inhibiting Mineralization of Natural Tissue DuringImplantation, and U.S. Pat. No. 5,215,541 (Nashef et al.) entitledSurfactant Treatment of Implantable Biological Tissue to InhibitCalcification. Recently a new technique of calcium mitigation by hightemperature fixation of the tissue in glutaraldehyde has been developedand was described in U.S. Pat. No. 5,931,969 (Carpentier et al.)entitled Methods And Apparatus For Treating Biological Tissue ToMitigate Calcification. Although some of these techniques have proven tobe efficient in reducing calcification, there remains a need in the artfor further improvements of the existing techniques or for thedevelopment of new calcification-mitigating techniques to lessen thepropensity for post-implantation calcification of fixed bioprosthetictissues.

SUMMARY OF THE INVENTION

The present invention provides methods for treating tissue to inhibitpost implant calcification whereby fixed, unfixed or partially fixedtissue is immersed in or otherwise contacted with a pre-treatedglutaraldehyde solution. In a preferred embodiment of the presentinvention, the glutaraldehyde solution is heat-treated prior to itscontact with the tissue. Preferably, the glutaraldehyde solution isheated to a first temperature for a first period of time. Thetemperature of the glutaraldehyde solution is then adjusted to a secondtemperature (preferably lower than the first temperature), beforecontacting the bioprosthetic tissue.

The first temperature to which the glutaraldehyde solution is heated issufficiently high, and is maintained for sufficiently long, to cause thefree aldehyde content and pH of the glutaraldehyde solution to fall by apredetermined amount. Preferably, the prior heat treating of theglutaraldehyde solution causes the free aldehyde concentration of thesolution to decrease by about 25%, preferably by about 50%. Theglutaraldehyde solution may be buffered so that the pH is initially inthe range of about 7.2 to 7.S, preferably about 7.4. After the heatinghas been carried out, the pH of the solution will typically have fallento approximately 5.0 to 7.0, preferably 6.0. Due to the preheating ofthe glutaraldehyde solution, the solution does not significantly changeits chemical characteristics when used to treat the tissue later in theprocedure.

In a preferred embodiment, the glutaraldehyde solution is heated to afirst temperature of at least 20° C., but preferably not more than 90°C. More preferably, the glutaraldehyde solution is heated to atemperature between about 60° C. to SO° C., and most preferably about70° C.±5° C. The glutaraldehyde solution may become somewhat yellow incolor during this heat-treatment step. The time period during which thefirst temperature must be maintained will typically vary inversely withthe first temperature (Le., lower temperatures will require a longerperiod of time to cause a decrease in free aldehyde content and/or afall in pH). Preferably, the glutaraldehyde is heated to the firsttemperature for a period of time between about one hour and six months,and more preferably about 1 day to 2 months. Thereafter, the solution isfiltered and adjusted to a second temperature before adding the tissue.Preferably, this second temperature may be in the range of about 30° C.to 70° C., preferably about 40-60° C., and more preferably about 50°C.±5° C.

In another embodiment of the present invention, glutaraldehyde solutionis not heat treated but the pH of the glutaraldehyde solution isadjusted to a pH within the range of about 5.0 to 7.0, and preferably toabout 6.0. The pretreated glutaraldehyde solution, whether by preheatingor pH adjustment, is then used to treat the tissue, preferably at atemperature in the range of about 30° C. to 70° C., more preferably at atemperature between about 40° C. to 60° C., and most preferably, at atemperature of about 50° C.±5° C. In a preferred embodiment, the tissueis treated for a period of time between about one hour to six months,and more preferably for about one day to two months. For example, at atemperature of about 50° C., the preferred period of time is betweenabout 5 days to 10 days, and most preferably, for about seven days.

The heat-treated or pH adjusted glutaraldehyde solution may, in somecases, also be used as a terminal sterilization solution such that thecalcification-decreasing treatment with previously treatedglutaraldehyde and a terminal sterilization may be carried outsimultaneously with the same solution and/or in a single container.

The heat-treated glutaraldehyde solutions may also contain otherchemicals to enhance its efficacy, such as surfactants (e.g., Tween®80), alcohol (e.g., ethanol) and/or aldehydes (e.g., formaldehyde).

In another embodiment of the method of the present invention, the tissueis heat treated in a preheated solution other than glutaraldehyde, forexample, any other fixative solution or a surfactant solution (e.g.,Tween® 80 with or without ethanol and/or formaldehyde), or a physiologicsolution (e.g., saline or a balanced salt solution). The preheating ofthe solution is carried out at a temperature between about 20° C. to 90°C., more preferably between about 37° C. and 60° C., and most preferablyabout 45° C., for one hour to six months, preferably one day to twomonths. In the preheated solution, the tissue is heat treated betweenabout 30° C. and 70° C., and more preferably about 50° C., for about oneday to two months. In another embodiment, the tissue is heat treated ina nonpreheat treated physiologic solution wherein the pH has beenadjusted between 5.0 and 7.0, preferably 6.0.

The method of the present invention results in a decrease in thetissue's propensity to calcify after being implanted within the body ofa human or animal patient. Prior to, concurrently with, or afterundergoing treatment with the pre-treated glutaraldehyde, the tissue maybe chemically fixed by exposing the tissue to one or more chemicalfixatives or cryopreserved by freezing the tissue in accordance withwell known techniques.

Further in accordance with the invention, there are providedbioprosthetic devices or articles that are formed, wholly or partially,of tissue that has been treated in accordance with the variousembodiments of the method of the present invention. Examples ofbiological tissues of human or animal origin which may be used inbioprosthetic devices or articles of the present invention include, butare not necessarily limited to: heart valves; venous valves; bloodvessels; ureter; tendon; dura mater; skin; pericardium; cartilage (e.g.,meniscus); ligament; bone; intestine (e.g., intestinal wall); smallintestinal submucosa (“SIS tissue”), and periostium.

Further in accordance with the present invention, there are providedmethods for treating diseases and disorders of mammalian patients, byimplanting bioprosthetic materials that have undergone the calcificationmitigating treatment of the various embodiments of the method of thepresent invention. Such treatment methods include, but are not limitedto, a) the surgical replacement of diseased heart valves withbioprosthetic heart valves that have been treated with glutaraldehyde inaccordance with the present invention, b) the repair or bypassing ofblood vessels by implanting biological vascular grafts that have beentreated with glutaraldehyde in accordance with the present invention, c)the surgical replacement or repair of torn or deficient ligaments byimplanting bioprosthetic ligaments that have been treated withglutaraldehyde in accordance with the present invention and, d) therepair, reconstruction, reformation, enhancement, bulking, ingrowth,reconstruction or regeneration of native tissues by implanting one ormore biopolymeric or bioprosthetic tissue scaffolds that have beentreated with glutaraldehyde in accordance with the present invention(e.g., tissue engineering with a natural tissue or biopolymericscaffold).

Still further in accordance with this invention, the various embodimentsof the method of mitigating post-implantation calcification ofbioprosthetic tissues offer significant advantages over previouspractices wherein glutaraldehyde was heated in the presence of thetissue, as the present invention allows the desirable features of theheat treatment to be achieved prior to any contact between theglutaraldehyde solution and the tissue, and also allows the temperatureof the glutaraldehyde solution to be lowered to about 30° C. to 70° C.,preferably about 40° C. to 60° C., or most preferably at about 50° C.prior to any contact with the tissue. This lessens the potential foruntoward or undesirable reactions to the bioprosthetic tissue due toexposure to high free aldehyde concentrations and/or long term heattreatment at temperatures above 60° C. It also allows for treatment ofthe tissue within realistic manufacturing time frames.

Still further in accordance with this invention, the method ofpreheating the solution, and/or heating the tissue, offer bettersterilization of both the solution and the tissue at the differentstages of the manufacturing process, including the terminal stage.Further aspects and advantages of the present invention will becomeapparent to those skilled in the relevant art, upon reading andunderstanding the “Description of Exemplary Embodiments” set forthherebelow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow diagram of one embodiment of the method for mitigatingcalcification of a bioprosthetic material, in accordance with thepresent invention.

FIG. 2 is a flow diagram of another embodiment of the method forpreparing a bioprosthetic device in accordance with the method of thepresent invention.

DESCRIPTION OF THE EXEMPLARY EMBODIMENTS

The following examples are provided for the purpose of describing andillustrating a few exemplary embodiments of the invention only. Oneskilled in the art will recognize that other embodiments of theinvention are possible, but are not described in detail here. Thus,these examples are not intended to limit the scope of the invention inany way.

It has previously been reported that cross-linked bioprosthetic tissuepost-treated in 0.625% glutaraldehyde phosphate solution for 2 months at50° C., with fluid movement (e.g., shaking), exhibited lesscalcification in the rat subcutaneous and rabbit intramuscular implantmodels than control cross-linked bioprosthetic tissue fixed in 0.625%glutaraldehyde phosphate solution under typical conditions (Le., roomtemperature for 1-14 days). See 66 Ann. Thoracic Surgery 264-6 (1998).Tissues treated under these conditions exhibited a characteristic tan tobrown appearance. The heated 0.625% glutaraldehyde phosphate solutionalso darkened to an amber-brown color and the aldehyde concentrationwithin that solution dropped to about 0.3%.

Since the above publication, the Applicant has discovered that it isadvantageous to conduct the heating step on the glutaraldehyde solutionprior to its contact with the tissue. The heat-treated glutaraldehydemay then be cooled to a lower temperature and the tissue may then beadded to the cooled glutaraldehyde solution under conditions of reducedseverity, greater convenience, or both (e.g., shorter time, lowertemperature, or both). By heat-treating the glutaraldehyde solution inthe absence of the tissue, higher temperatures, concentrations or bothcan be used during the heat-treating process without risking or causingany adverse effect on the tissue. In another embodiment, theglutaraldehyde solution can be buffered by adjusting the pH of thesolution to within a range of about 5.0 to 7.0, preferably about 6.0.Applicants have found that the buffered glutaraldehyde solution has asimilar, although slightly less, advantageous effect as the heat-treatedglutaraldehyde solution.

The mechanism by which the heat-treated glutaraldehyde mitigatespost-implantation calcification is not presently known with certainty.However, Applicants postulate that this calcification mitigating effectis due at least in part to the leaching of lipoproteins and calciumbinding proteins and in part to the formation of a calcificationmitigating chemical or moiety within the glutaraldehyde solution thatacts to limit or inhibit the fixation of calcium into the tissue, eitherby way of a physical barrier effect (i.e., by retarding diffusion at theboundary layer) and/or by chemically modifying the structure and thesurface charge of the tissue and thus its affinity to attract calciumions. Heat-treated glutaraldehyde can also be used to enhancesterilization by leaving the tissue in the heat-treated glutaraldehydeor by heating the tissue within the previously heat treatedglutaraldehyde solution to temperatures between about 37° C. and 60° C.

A. General Method for Mitigating Calcification of Bioprosthetic Material

FIG. 1 is a flow diagram that generally illustrates one embodiment ofthe method of the present invention. As shown in FIG. 1, the first stepof the process is to heat treat glutaraldehyde solution in the absenceof tissue. It will be appreciated that the concentration ofglutaraldehyde in the starting solution may be varied. Thereafter, thesolution concentration may be adjusted, if desired, prior to addition ofthe tissue. It is believed that glutaraldehyde concentrations of aslittle as 0.1% and as much as 25% or more may be used during theheat-treating step. Reduced glutaraldehyde concentrations of 0.6% to2.5% have, to date, been successfully obtained and used by Applicant,and those skilled in the art will recognize that higher or lowerconcentrations of glutaraldehyde may indeed prove to be advantageousduring the heat-treating step of the process. The preferredconcentration for use during the heat-treating step (FIG. 1) is1.0-2.0%. This heat-treating of the glutaraldehyde may be accomplishedby heating of the solution until the free aldehyde content of thesolution has fallen about 2S % or more and remains stable at that level(e.g., a solution of 1.8% falls to about 0.6% or less). Initially, thesolution containing glutaraldehyde may be buffered to a pH of 7.4 with aphosphate buffer, a non-phosphate buffer such as a HEPES buffer, orother suitable buffered solutions, and, in such cases, heating of thesolution to cause the free aldehyde content to fall will also cause thepH of the solution to fall. In another embodiment of the presentinvention, rather than heat treating the glutaraldehyde solution, the pHmay be adjusted from 7.4 to a pH within the range of about 5.0 to 7.0,preferably 6.0.

The heat-treating of the glutaraldehyde may be accomplished by anysuitable means. In this example, the glutaraldehyde is pre-heated to andmaintained at a temperature between about 20-90° C., preferably betweenabout 60° C.-80° C., and most preferably 70° C.±5° C. for a sufficientperiod of time to cause the free aldehyde concentration to decrease byat least 2S % and to stabilize at a pH of approximately 6.0 (i.e., thepH of 6.0 corresponds to a free aldehyde concentration of about0.3-0.7%). Depending on the temperature used, the step of heat treatingthe glutaraldehyde may take anywhere from one hour to six months or moredepending on the temperature used. The preferred method is to heat theglutaraldehyde solution to approximately 70° C.±5° C., for approximately1 day to 2 months or until the desired fall of at least 2S % or more infree aldehyde concentration and a pH of approximately 6.0, are observed.

After the heat-treatment of the glutaraldehyde has been completed thesolution is cooled to a second temperature that does not cause damage tothe tissue (e.g., about 30° C. to 70° C., preferably about 40° C. to 60°C., or most preferably at about SO° C.). An unfixed, partially-fixed, orfixed tissue is then contacted with the heat-treated glutaraldehyde.Tissue that has been “fully fixed” in this regard means that the tissuehas been fixed to an extent suitable for use as an implant, while“partially fixed” means that the tissue has been fixed to some extentshort of being fully fixed. This tissue treatment step is preferablyaccomplished by immersing fixed, partially fixed or unfixed tissue inthe heat-treated glutaraldehyde solution while maintaining the solutionat about 30° C. to 70° C., preferably about 40° C. to 60° C., or mostpreferably at about 50° C. It is preferable that the pH of the solutionbe left at about 6.0 prior to placement of the tissue within thesolution. Thereafter, the temperature of the solution is maintained atapproximately 50° C. with the tissue immersed in the solution to allowthe heat-treated glutaraldehyde solution to interact with or modify thetissue. The tissue's susceptibility to post-implant calcification willbe significantly reduced after immersion for as little as one hour to asmuch as six months or more (depending primarily on the temperatureused), but typically occurs within 1 to 15 days at 50° C.

In another embodiment of the method of the present invention, the tissuemay be heat treated in a surfactant solution (e.g., Tween® 80 with orwithout ethanol and/or formaldehyde) or in a physiologic solution (e.g.saline or a balanced salt solution) at a temperature between about 37°C. and 60° C., preferably about 45° C., for about one hour to sixmonths, preferably about one to 15 days, and then heat treated in aglutaraldehyde solution as described above.

Prior to, concurrently with or after the tissue treatment step, thetissue may be cryopreserved or otherwise preserved, i.e. by fixation.

B. An Example of a Method for Manufacturing a Fixed Heterologous HeartValve Bioprosthesis Having Mitigated Propensity for Post-ImplantationCalcification

FIG. 2 is a flow diagram of a specific process for manufacturing abioprosthetic device, such as a stented or stentless porcine heart valveor bovine pericardial heart valve of the type referred to herein. Thefollowing is a description of the exemplary process shown in FIG. 2.

1. Heat-Treating of Glutaraldehyde

Prepare Glutaraldehyde Solution

Initially, an aqueous solution of 1.8% by weight glutaraldehyde isprepared in a clean, inert vessel (e.g., a vessel made of stainlesssteel, plastic or borosilicate glass) and such solution is then bufferedto the pH of approximately 7.4 by adding phosphate buffered salinesolution.

Preheat Glutaraldehyde Solution in Absence of Tissue

The glutaraldehyde in the solution is then preheated. Such preheating ofthe glutaraldehyde is accomplished by heating of the solution to about70° C.±SoC and maintaining such temperature until the pH of the solutionfalls to approximately 6.0. At this point, the color of the solution canbe colorless to golden or brown. The fall of the solution pH to 6.0 andthe accompanying change in color to golden or brown indicates that thepreheating treatment has been completed. This preheating step istypically completed after 1-14 days, preferably 6-8 days, of maintainingthe solution at the 70° C.±5° C. temperature. Higher temperaturesranging up to approximately 90° C. may be used, and the use of suchhigher temperatures will typically speed the desired fall in freealdehyde concentration and accompanying change in pH (e.g., a solutionhaving a starting pH adjusted to 7.4 will fall to a pH of about 6.0after approximately 1-3 days at 90° C.). Lower temperatures, rangingdownward to approximately 20° C., may also be used, and the use of suchlower temperatures will typically cause the desired free aldehydecontent and pH changes to take longer. After the heat treatment of thesolution has been carried out the solution is filtered.

Optional Neutralization of pH of Heat-Treated Glutaraldehyde Solution

After the glutaraldehyde has been heat-treated, the solution is allowedto cool to about SO° C. and its pH may be adjusted at step 24 back toapproximately 7.4 by adding phosphate buffered saline or some othersuitable buffer.

2. Harvesting, Preparation and Fixation of Tissue:

Harvesting/Preparation of Biological Tissue

The desired biological tissue is harvested from a human cadaver oranimal donor, and prepared for subsequent fixation and treatment. Thetissue is typically harvested by surgical cutting or removal from itshost animal. Thereafter, it is typically trimmed or cut to size andwashed with sterile water, basic salt solution, saline or other suitablewashing solution.

Fixation of Biological Tissue

The biological tissue may be fixed prior to, during or after itstreatment with the heat-treated glutaraldehyde. In this example, thetissue is fixed prior to undergoing the treatment with heat-treatedglutaraldehyde. This fixation is carried out by immersing the tissue ina solution of 0.625% by weight glutaraldehyde buffered to a pH ofapproximately 7.4 by a suitable buffer such as a phosphate buffer, for1-14 days at ambient temperature. In order to enhance fixation orsterilization other chemical compounds such as surfactants (e.g. Tween®80) and/or ethanol and/or formaldehyde can be added to theglutaraldehyde. It will be appreciated, however, that various otherfixatives may be used, such as aldehydes (e.g., formaldehyde,glutaraldehyde, dialdehyde starch) or polyglycidyl ethers (e.g., Denacol810), or heterologous bifunctional or multifunctional crosslinkers.

Rinsing of Tissue

After it has been removed from the fixative solution, the tissue isthoroughly rinsed with saline solution, basic salt solution or freeglutaraldehyde solution or some other suitable washing solution.

3. Treatment of Tissue with Heat-Treated Glutaraldehyde to MitigatePost-Implantation Calcification:

Immersion of Tissue in Heat-Treated Glutaraldehyde Solution

After the fixed tissue has been rinsed, it is treated with the pre-heattreated glutaraldehyde solution. The pre-heat treated glutaraldehydesolution is placed in a vessel such as a stainless steel bath, cooled toand maintained at preferably 50° C.±5° C. The fixed/rinsed tissue isthen immersed in the heat-treated glutaraldehyde solution and thesolution is continually maintained at 50° C.±5° C. with the tissueimmersed in the solution with or without fluid movement. The tissue'ssusceptibility to post-implant calcification will be significantlyreduced after immersion for as little as one hour to as much as sixmonths or more (depending primarily on the temperature used), buttypically occurs within 6 to 8 days at 50° C.±5°. Thereafter, the tissueis removed from the solution. The tissue is typically brown in color atthis time.

Rinsing of Tissue

After it has been removed from the heat-treated glutaraldehyde solution,the tissue is thoroughly rinsed with saline solution, basic saltsolution or some other suitable washing solution.

4. Poststerilization, Assembly/Fabrication and Storage of Bioprosthesis

First Bioburden Reduction (BREP I)

After the tissue has been fixed, treated with the heat-treatedglutaraldehyde and rinsed, it is subjected to a first bioburdenreduction treatment immersed in or otherwise contacted with a mixturecontaining i) a crosslinking agent, ii) a denaturing agent and iii) asurfactant (i.e., a CDS solution). One preferred CDS solution (describedin U.S. Pat. No. 4,885,005 and U.S. Pat. No. 4,648,881) is a mixture ofi) formaldehyde, ii) ethanol and ii) surfactant (e.g., Tween® 80surfactant, available from ICI Americas, Brantford, Ontario). Suchpreferred CDS solution may also be referred to by the acronym “FETS” andhas a preferred formulation as follows:

-   Formaldehyde 4.0.±.0.4% by weight-   Ethanol 22.0.±.2.2% by weight-   Tween® 80 1.2.±.0.2% by weight

The tissue is preferably immersed in the CDS solution for 2 hours to 7days and typically about 2 hours. During this immersion period, the CDSsolution is maintained at a temperature of 4-50° C., and preferably atabout 20-37° C.

Those skilled in the art will appreciate that various alternativechemical compounds or solutions may be substituted for each component ofthe CDS solution, as follows:

Potential Alternative Crosslinking Agents:

A. Aldehydes: formaldehyde, glutaraldehyde, paraformaldehyde,glyceraldehyde, glyoxal acetaldehyde or acrolein

B. Epoxides: any of the various Denacols and their individual reactivespecies, including mono, di, tri, and multi-functionalized epoxides

C. Carbodiimides

D. Mixed multifunctional molecules (e.g. aldehyde-epoxide combination)

Potential Alternative Denaturing Agents:

A. Alcohols/Solvents: e.g., ethanol, isopropyl alcohol

B. Acidified Ethers: e.g., sulfuric acid/ether mixture, acetone, ethersof small alkyl size (methyl, ethyl, etc. but probably not beyond butyl)

C. Ketones: e.g., methyl ethyl ketone (MEK)

D. Commercial Solvent Systems: e.g., Genesolve™ (Allied Signal, Inc.,Morristown, N.J.)

E. Glycols: glycerol ethylene glycol, polyethylene glycol, low molecularweight carbowax

F. Chaotropic Agents: e.g., urea, guanidine hydrochloride, guanidinethiocyanate, potassium iodide

G. High Concentration Salt Solutions: e.g., lithium chloride, sodiumchloride, cesium chloride.

Potential Alternative Surfactants:

(these surfactant compounds can be used individually or in mixtures suchas deoxycholate/Triton or commercially-available mixtures such asMicro-80/90.)

A. Anionic Surfactants: e.g., esters of lauric acid, including but notlimited to sodium laurel sulfate (also called sodium dodecyl sulfate)

B. Alkyl sulfonic acid salts: e.g., 1-decanesulfonic acid sodium salt

C. Non-ionic compounds: e.g., compounds based on the polyoxyethyleneether structures, including Triton X-IOO, 114, 405, N-101 (availablecommercially from Sigma Chemical, St. Louis, Mo.) and relatedstructures; Pluronic and Tetronic surfactants (available commerciallyfrom BASF Chemicals, Mount Olive, N.J.)

D. Alkylated Phenoxypolyethoxy Alcohols: e.g., NP40, Nonidet P40,Igepal, CA630, hydrolyzedlfunctionalized animal and plant compoundsincluding Tween® 80, Tween® 20, octyl-derivatives, octyl b-glucoside,octyl bthioglucopyranoside, deoxycholate and derivatives thereof,zwitterionic compounds, 3-([cholamidopropyl]-dimethylamino)-1-propanesulfonate (CHAPS), 3-([cholamidopropyl]-dimethylamino)-2-hydroxy-1-propanesulfonat-e (CHAPSO) (available from PierceBiotec Company, Rockford, Ill.).

Fabrication/Assembly

After the first bioburden reduction has been completed, the tissue maybeagain rinsed with a suitable rinsing solution such as isotonic saline or0.625% glutaraldehyde and transported into a clean room or asepticenvironment. Thereafter, the tissue may be further trimmed or shaped (ifnecessary) and attached to or assembled with any non-biologicalcomponents (e.g., stents, frames, suture rings, conduits, segments ofpolyester mesh to prevent suture tear-through, etc.) to form the desiredbioprosthetic device. Examples of bioprosthetic devices that areassembled of both biological tissue and non-biological componentsinclude stented porcine bioprosthetic heart valves (e.g., theCarpentier-Edwards® Bioprosthesis), and bovine pericardial heart valves(e.g., Carpentier-Edwards® Pericardial Bioprosthesis), stentless porcineaortic valves that incorporate fabric reinforcements (e.g., EdwardsPRIMA Plus® Stentless Aortic Bioprosthesis), and conduit valves forbio-mechanical ventricular assist devices (e.g., the Novacor N-100PCmodel), all available from Edwards Lifesciences LLC, Irvine, Calif.

Second Bioburden Reduction (BREP II)

After the bioprosthesis has been fabricated and assembled it issubjected to a second bioburden reduction that is essentially a repeatof the first bioburden reduction described above, however, in thissecond bioburden reduction step, the solution is preferably maintainedat about 37° C. for approximately 2 hours to 10 days, preferably about 9hours.

Terminal Heating and Storage

After completion of the second bioburden reduction, the tissue (orbioprosthesis) is rinsed with a suitable rinsing solution (such asisotonic saline or 0.625% glutaraldehyde solution) and then” placed in aterminal solution for storage and sterilization. The preferred terminalsolution is a glutaraldehyde solution having a concentration of about0.2 to 1.0% by weight glutaraldehyde, and most preferably about 0.625%by weight glutaraldehyde. This solution has a strong sterilizing effectthat can be enhanced by a terminal heating of the solution.

In this terminal sterilization step, the tissue (or bioprosthesis) isimmersed in or contacted with the terminal solution and heated for aperiod of time sufficient to ensure sterility of the bioprosthesis untilthe time of implantation. The period of heating varies depending uponthe temperature utilized, i.e., the lower the temperature the longer theperiod of time. For example, from 1 or 2 hours to 1 month fortemperatures between about 50° C. and 20° C., respectively. Preferably,the period of time is 1 to 6 days at 37° C. or 6 hours to 2 days at 50°C., but one of skill in the art will recognize that these temperature ortime values can be modified within the scope of the invention.

In order to avoid additional transfer and manipulation, the terminalheating is preferably carried out in the sealed storage container orpackage in which the bioprosthesis will be shipped and stored until thetime of implantation. The tissue (or bioprosthesis) is asepticallydeposited in the storage container that has been pre-filled with the0.625% glutaraldehyde aqueous solution buffered to a pH of 7.4 withsodium hydroxide, such that the tissue (or bioprosthesis) is fullyimmersed in the buffered glutaraldehyde solution. Thereafter, thecontainer is sealed and placed at room temperature for at least 7 days,or in an oven at 37° C. for 24 hours, or at 50° C. for 6 hours toenhance the sterilization power of glutaraldehyde. Thereafter, thecontainer is cooled to room temperature and shipped to the hospital orother location(s) where it is stored until the time of use of thebioprosthesis.

In another embodiment, the terminal heating is carried out beforeplacing the tissue or bioprosthesis in the storage container.

In some cases, glutaraldehyde that has been heat-treated in accordancewith this invention may be used as the terminal solution and, in suchcases, it may be possible to shorten or completely eliminate theprevious step of immersing the tissue in previously heat-treatedglutaraldehyde, opting instead to accomplish some or all of thetreatment of the tissue by heat-treated glutaraldehyde until the laststep of storage, i.e., concurrently with the terminal sterilizationstep.

While the foregoing is a complete description of the preferredembodiments of the invention, various alternatives, modifications, andequivalents may be used. Moreover, it will be obvious that certain othermodifications may be practiced within the scope of the appended claims.

What is claimed is:
 1. A method for mitigating post-implantationcalcification of a bioprosthetic implant tissue, said method comprisingthe steps of: obtaining biological tissue; exposing the tissue to afixation process to form cross-linkages in the tissue to obtain a fixedtissue; providing a starting solution selected from the group consistingof a glutaraldehyde solution and a physiologic solution; adjusting thepH of the starting solution to a pH of 6.0 without heating the startingsolution; immersing a quantity of the fixed tissue in the pH-adjustedsolution; and heating the pH-adjusted solution and the fixed tissue to aheat treating temperature in the range of about 30° C. to 70° C. for aperiod of time.
 2. The method of claim 1, wherein the heat treatingtemperature is in the range of about 40° C. to 60° C.
 3. The method ofclaim 1, wherein the heat treating temperature is about 50° C.±5° C. 4.The method of claim 1, wherein the period of time is between about onehour and six months.
 5. The method of claim 1, wherein the period oftime is between about one day and two months.
 6. The method of claim 1,wherein the period of time is between about 5 days to 10 days.
 7. Themethod of claim 1, wherein the period of time is about 7 days.
 8. Themethod of claim 1, wherein the starting solution is aqueousglutaraldehyde buffered to a starting pH, wherein the step of adjustingcomprises lowering the pH.
 9. The method of claim 8, wherein thestarting pH is between about 7.2 to 7.8.
 10. The method of claim 1,wherein the bioprosthetic implant material is a bioprosthetic heartvalve, and the method further comprises the steps of: harvesting thebiological tissue from a human cadaver or animal donor; preparing thetissue by trimming to size and washing with a suitable washing solution;and assembling the heat treated and fixed tissue with non-biologicalcomponents to form a bioprosthetic heart valve.
 11. The method of claim10, wherein the heat treating temperature is in the range of about 40°C. to 60° C.
 12. The method of claim 10, wherein the heat treatingtemperature is about 50° C.±5° C.
 13. The method of claim 10, whereinthe period of time is between about one day and two months.
 14. Themethod of claim 10, wherein the period of time is between about 5 daysto 10 days.
 15. The method of claim 10, wherein the period of time isabout 7 days.
 16. The method of claim 10, further including, after thetissue has been fixed and heat treated, and prior to assembly into aheart valve, subjecting the fixed tissue to a first bioburden reductionprocess including: contacting the fixed tissue with a mixturecontaining: i) a crosslinking agent, ii) a denaturing agent and iii) asurfactant.
 17. The method of claim 16, further including, afterassembling the heart valve, subjecting the fixed tissue to a secondbioburden reduction process including: contacting the fixed tissue witha mixture containing: i) a crosslinking agent, ii) a denaturing agentand iii) a surfactant.
 18. The method of claim 17, wherein the secondbioburden reduction process is done at a temperature of about 37° C. fora period of time longer than the first bioburden reduction process. 19.The method of claim 18, further including storing the heart valve in asolution of glutaraldehyde.
 20. The method of claim 10, wherein thestarting solution is aqueous glutaraldehyde buffered to a starting pH,wherein the step of adjusting comprises lowering the pH.
 21. The methodof claim 20, wherein the starting pH is between about 7.2 to 7.8.